U.S. patent number 7,785,770 [Application Number 11/444,819] was granted by the patent office on 2010-08-31 for sugar-containing hydrogel for immobilization.
This patent grant is currently assigned to The United States of America as represented by the Secretary of the Navy. Invention is credited to Paul T. Charles, Brett D. Martin, Charles H. Patterson, Jr., Mark S. Spector, David A. Stenger.
United States Patent |
7,785,770 |
Spector , et al. |
August 31, 2010 |
Sugar-containing hydrogel for immobilization
Abstract
The use of sugar-containing hydrogels as very highly porous,
aqueous support material for the immobilization of
oligonucleotides, peptides, proteins, antigens, antibodies,
polysaccharides, and other biomolecules for sensor applications.
Unusually large sizes of interconnected pores allow large target
molecules to pass rapidly into and through the gel and bind to
immobilized biomolecules. Sugar-containing hydrogels have extremely
low non-specific absorption of labeled target molecules, providing
low background levels. Some hydrogel materials do not have this
type of homogeneous interconnected macroporosity, thus large target
molecules cannot readily diffuse through them. Additionally, they
nearly always experience non-specific absorption of labeled target
molecules, limiting their usefulness in sensor applications. A
method is provided for preparing sugar polyacrylate hydrogels with
functional chemical groups which covalently bond oligonucleotides
and peptides. A method for copolymerizing acrylate-terminated
oligonucleotides with sugar acrylate monomers and diacrylate
cross-linking agents is also provided.
Inventors: |
Spector; Mark S. (Springfield,
VA), Stenger; David A. (Herndon, VA), Patterson, Jr.;
Charles H. (Glen Burnie, MD), Martin; Brett D.
(Washington, DC), Charles; Paul T. (Bowie, MD) |
Assignee: |
The United States of America as
represented by the Secretary of the Navy (Washington,
DC)
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Family
ID: |
34080577 |
Appl.
No.: |
11/444,819 |
Filed: |
May 19, 2006 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20060246499 A1 |
Nov 2, 2006 |
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Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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10627143 |
Jul 25, 2003 |
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Current U.S.
Class: |
435/4; 526/89;
526/72; 530/350; 435/287.2; 435/14; 530/300; 435/6.11 |
Current CPC
Class: |
G01N
33/54353 (20130101); G01N 33/548 (20130101) |
Current International
Class: |
C12Q
1/00 (20060101) |
Field of
Search: |
;435/4,6,287.2,7.1,34,14,8 ;526/72,89 ;530/300 |
References Cited
[Referenced By]
U.S. Patent Documents
Other References
Merriam-Webster Dictionary On-line;
www.merriam-webster.com/dictionary/Nucleic%20Acid downloaded on
Dec. 13, 2009; 2 pages. cited by examiner .
Rethwisch et al. Materials Research Soc. Symposium Proceedings
(1994) 320(Biomoleulcar Materials by Design); 225-230. cited by
examiner.
|
Primary Examiner: Marx; Irene
Assistant Examiner: Hanley; Susan
Attorney, Agent or Firm: Ressing; Amy Hunnius; Stephen
T.
Parent Case Text
This application is a divisional application of U.S. application
Ser. No. 10/627,143 filed on Jul. 25, 2003, incorporated herein by
reference.
Claims
The invention claimed is:
1. A method for assaying biomolecules wherein said assay is
selected from the group consisting of a fluorescence assay, a
radioactive assay, a magnetic assay and an optical assay,
comprising the steps of: (A) functionalizing a support with
acrylate groups; (B) reacting 6-acryloyl-beta-O-methyl
galactopyranoside, a crosslinker selected from the group consisting
of a bis-acrylamide, a bis-acrylate, and a bis-vinyl and
2-acrylamido hydroxyacetic acid to form a polyacrylate hydrogel;
(C) reacting said polyacrylate hydrogel with said acrylate groups
of said support to form a polyacrylate hydrogel linked to the
support; (D) reacting said biomolecule to be assayed with said
polyacrylate hydrogel linked to said support to form a covalent
bond between said biomolecule and said hydrogel, and (D) assaying
said covalently bonded biomolecule.
2. The method according to claim 1, wherein said biomolecule is
selected from the group consisting of a DNA comprising up to
100,000 nucleotide base units and a protein.
3. The method according to claim 1, wherein said biomolecule
comprises a fluorophore group.
4. The method according to claim 1, wherein said biomolecule is
CY3-Staphylococcal enterotoxin B (SEB).
5. A method for making a hydrogel polymer comprising the steps of:
polymerizing 6-acryloyl-beta-O-methylgalactopyranoside, a
crosslinker with two or more polymerizable double bonds, an
acrylate-substituted oligonucleotide and a compound having a group
selected from the group consisting of an amino group and a carboxyl
group, wherein said crosslinker is selected from the group
consisting of bis-acrylamide, bis-acrylate, and bis-vinyl
compounds; and forming a hydrogel polymer having a pore size of
from 0.1 to 0.6 microns or greater than 2 microns.
Description
BACKGROUND OF THE INVENTION
Field and Background of the Invention
Immobilization of deoxyribonucleic acid (DNA), ribonucleic acid
(RNA), proteins, antigens, and other biomolecules on a variety of
solid substrates, typically glass, provides the basis for
array-based bioassays. Examples of such technologies include
patterning of DNA probes in hybridization assays for clinical
diagnostics, drug discovery, and pathogen detection and arraying
proteins and antigens for antibody detection. A number of
strategies have been developed for the attachment of oligomers to
glass substrates. Single-stranded DNA (ssDNA) probes are commonly
synthesized on surfaces photolithographically, Pease et al, Proc.
Natl. Acad. Sci. USA. 1994, 91, 5022-5026, electrostatically
adsorbed to the substrate, Schena et al, Science 1995, 270, 467470
or covalently attached to a self-assembled monolayer, Chrisey et
al, Nucleic Acid Res. 1996, 24, 3031-3039, Zammatteo et al. Anal
Biochem. 2000, 280, 143-150,
A major limitation for the use of microarrays in pathogen detection
is the low signal levels observed when probe DNA is confined to the
substrate surface. An alternative is immobilizing ssDNA in a
three-dimensional hydrogel allowing for higher density and
sensitivity, Timofeev et al, Nucleic Acid Res. 1996, 24, 3142-3149.
U.S. Pat. No. 5,981,734 describes a method for immobilizing
biomolecules such as oligonucleotides in polyacrylamide gels,
either through copolymerization of allyl-substituted
oligonucleotides or incorporation of functional groups which can be
covalently coupled to modified oligonucleotides. U.S. Pat. No.
6,180,770 describes a method for preparing oligonucleotides
derivatized with a terminal polymerizable acrylamide. These
monomers can then be copolymerized into an acrylamide hydrogel to
produce a polymerized layer containing the covalently linked ssDNA
molecule. This technology has been licensed to Apogent Discoveries
and is commercially available.
Conventional hydrogels suffer from a number of limitations. In
general, it is difficult to obtain water contents greater than 95 w
%. This results in a small mesh size in the gel, limiting the
diffusion of large macromolecules or particles. For example, target
ssDNA with length greater than 200 nucleotides are unable to
permeate into a standard 5% polyacrylamide gel, Guschin et al,
Anal. Biochem. 1997, 250, 203-211. The networks are often
heterogeneous and the monomers can be toxic (e.g. acrylamide). The
polymeric hydrogels described in this Disclosure, for example those
based on monomeric sugar acrylates or methacrylates, do not
experience the drawbacks outlined above. Enzymatic acryloylation
provides a simple method for forming the monomers with high
regioselectivity, Martin et al, Macromolecules, 1992, 25,
7081-7085. These hydrogels have equilibrium water contents above
95% resulting in pore sizes of 500 nm or larger, Martin et al,
Biomaterials, 1998, 19, 69-76. U.S. Pat. No. 5,854,030 provides the
methodology for the chemoenzymatic synthesis of the monomers and
subsequent formation of the hydrogels. The above cited references,
including publications and patents are incorporated herein by
reference in their entirety.
SUMMARY OF THE INVENTION
This invention details the synthesis of polymeric sugar-containing
hydrogels and their use as three-dimensional, highly macroporous
substrates for the immobilization of oligonucleotides, peptides,
proteins, and other biomolecules. These hydrogels are formed from
compounds containing polymerizable double bonds. Examples of such
compounds include, but are not limited to, acrylates,
methacrylates, acrylamides and methacrylamides. The sugar compounds
may be hexose, pentose, tetrose, or triose, or monosaccharide, di-,
tri-, tetra-, penta-, hexa-, hepta-, octa-, nona-, or
decasaccharides. If glycosides are used, they may contain either
alpha or beta aglycon linkages. The hydrogel provides a support
with activated functional groups for biomolecule attachment
throughout the matrix The high porosity of the sugar-containing
hydrogels allows for rapid diffusion of large (up to two micron
diameter) molecules or particles. This includes long DNA sequences
(e.g. greater than 100,000 nucleotide bases) and large antibodies,
functionalized microbeads as well as semiconductor and metal
nanoparticles currently being explored as alternatives to
conventional fluorophores for ultrasensitive optical detection. A
further advantage of the hydrogel matrix is its extremely low
nonspecific absorption of labeled biomolecular targets, and the
large number of reactive sites available for molecular attachment.
The high density of immobilized probes throughout the volume of the
gel leads to a greater detection sensitivity versus a similarly
derivatized flat solid substrate.
Three methods for incorporating biomolecules into the
sugar-containing hydrogels are disclosed. All methods result in
covalent linkage of the biomolecules to the three-dimensional gel
matrix. In the first case, oligonucleotides with a terminal
acrydine unit are polymerized with a sugar compound having a
polymerizable double bond such as a sugar acrylate or sugar
methacrylate monomer and a crosslinker having at least two
polymerizable double bonds, providing a direct covalent link to the
acrylate backbone.
In the second case, a sugar compound having a polymerizable double
bond such as a sugar acrylate or sugar methacrylate monomer is
polymerized with a crosslinker having at least two polymerizable
double bonds and a third compound having a polymerizable double
bond and a group selected to allow covalent attachment of
oligonucleotides, peptides, proteins, or other biomolecules. The
crosslinker and the third compound may contain an acrylate,
methacrylate, acrylamide, or methacrylamide moiety. In one
instance, amino groups are introduced into the gel by using
N-(3-aminopropyl) methacrylamide as a monomer. A number of
strategies are then available for the attachment of biomolecule
amino groups to the gel polymer. Aldehyde terminated
oligonucleotides, peptides, proteins, or other biomolecules react
with the amine in the presence of a reducing agent, forming a
covalent bond Phosphorylated or carboxylated oligonucleotides,
peptides, proteins, or other biomolecules can be covalently
attached to the amino group using carbodiimide condensation
mediated by a compound such as EDC. Amino terminated
oligonucletides, peptides, proteins, or other biomolecules can be
coupled using a homobifunctional crosslinker such as
diisothiocyanate or bis(sulfosuccinimidyl) suberate (BS.sup.3). In
the third instance, carboxyl groups are introduced into the gel by
introducing N-(3-carboxylpropyl) methacrylamide as a termonomer.
Amino terminated oligonucleotides can be covalently attached to the
carboxyl group through carbodiimide condensation. In a final
instance, aldehyde groups are introduced into the gel by using
N-(5,6-di-isopropylidene) hexylacrylamide as a termonomer.
Aldehydes can then be generated by removing the isopropylidene
protecting groups using acetic acid (Timofeev et al, 1996).
Aminated oligonucleotides, peptides, proteins, or other
biomolecules can then be reacted with the aldehyde groups, forming
covalent linkages. The gels described in this Invention have water
contents of at least 90 wt %, and in preferred embodiments have
water contents of 94 wt % of greater.
FIGURES
FIG. 1. shows one possible generalized chemical structure of the
polymer network component of the sugar-containing hydrogen of this
invention. In the preferred embodiments, R.sub.1 is H, alkyl or
phenyl, R.sub.2-R.sub.7 are H, OH, O-phenyl, or O-methyl, R.sub.5
is H or methyl, R.sub.9 is OH, propane 1,3 diamine, or aminohydroxy
acetic acid, and R.sub.10 is H or methyl. R.sub.9 can also be a
biomolecule covalently attached via an amine linkage. In this
Figure the repeat units m, n, and p are residues originating from
acrylate, methacrylate, acrylamide, or methacrylamide monomers.
FIG. 2 shows the chemical structures of several carbon-carbon
double-bond containing bis-crosslinkers that could be used to form
the polymer network.
FIG. 3 shows the structure of two crosslinkers used to attach DNA,
peptides, proteins, or other biomolecules via amine
linkages--BS.sup.3 (top) and EDC (bottom).
FIG. 4. is a reaction diagram showing an EDC-mediated activation of
a carboxylate moiety in the gel polymer network, and further
reaction of the activated moiety with an amino group of an
oligonucleotide, peptide, protein, or other biomolecule resulting
in covalent attachment of the latter.
FIG. 5. demonstrates that there is very little non-specific bonding
of target molecueles to the sugar hydrogels of this invention.
FIG. 6. describes the time-dependent movement of fluorescent
2-micron diameter beads through an unmodified sugar poly(acrylate)
hydrogel. Open circles, experimental data; filled circles,
diffusion theory.
FIG. 7. shows a micro array formed using the reaction an amino
terminated DNA labeled in the 5' end with a florophore (Cy3) with
an activated amino sugar hydrogel of the invention on a
support.
FIG. 8. shows a micro array of the DNA of FIG. 4 after reaction
with a carboxylate-modified sugar hydrogel on a support.
FIG. 9. shows a micro array of a fluorophore labeled protein
coupled to a carboxylate modified sugar hydrogel on a support.
EXPERIMENTAL RESULTS
The galactose acrylate monomer, 6-acryloyl-.beta.-O-methyl
galactopyranoside (1) where R.sub.1 is CH3 was chemoenymatically
prepared using the procedure of Martin et al, 1992. The lipase from
Pseudomonas cepacia catalyses the regioselective acryloylation at
the 6-hydroxi moiety of .beta.-O-methyl galactopyranoside in
anhydrous pyridine to give the monoacrylate. The acrylate (1)
exists as .alpha. and .beta. anomers and either or both may be used
to create the sugar hydrogels of this invention.
##STR00001##
In structure (1) and all sugar acrylates or sugar methacrylates
used in this invention, R.sub.1 is preferably a methyl group,
R.sub.2-R.sub.7 are preferably H or OH For sugar acrylate, R.sub.8
is H; for sugar methacrylate, R.sub.8 is methyl. However, R.sub.1
may also be without limitation, H, alkyl, aromatic, carbohydrate,
and acryl and acrylamido. R.sub.2-R.sub.7 may be in addition to H,
or OH, isopropyl, alkyl, aromatic. It should be understood that
other groups may be selected for R.sub.1, R.sub.2, and R.sub.7
without deviating from the bounds of this invention. The sugar
compounds (1) of this invention may be mono, di, or
polysaccharides.
One possible generalized polymeric structure of the gel described
in this invention is shown in FIG. 1. In this the sugar acrylate or
methacrylate (1) of choice is polymerized with a multifunctional
bis-crosslinker having at least two polymerizable double bonds and
a third compound having a polymerizable double bond and an amine,
carboxyl or other group capable of forming covalent bonds with
oligonucleotides and/or proteins. The crosslinking compounds are
selected from bis-acrylamides, bis-acrylates and bis-vinyl
compounds (FIG. 2). The third compound is selected so that when the
sugar hydrogel polymer is formed, the amino or carboxyl groups of
the third compound provide reactive sites on the polymer backbone
for reaction with coupling agents (FIG. 3) that allow covalent
attachment of oligonucleotides and proteins and other biomolecules
of interest. Covalent bonding of the polymer reactive sites with
compounds of interest provides the basis of assay for the target
molecules of interest
Copolymerization of Acrydine DNA with Sugar Acrylate
Oligonucleotides containing an acrylic acid group directly attached
to their 5'-end were purchased from Integrated DNA Technologies.
Samples were prepared on glass slides that had been functionalized
with methacrylate groups using the following procedure. The glass
slide is cleaned a by immersion in a hydrochloric acid/methanol
mixture, followed by sulfuric acid and treated with a 4% (v/v)
solution of methacryloxypropyl trimethoxysilane (MTPTS) (93 mL
methanol, 2.7 mL water, 0.3 mL glacial acetic acid, 4 mL of silane)
at 60.degree. C. for 1 hour. The slides are then rinsed in
methanol, water, and methanol again. The slides are baked for 5
minutes at 120.degree. C. Slides can be stored in a dessicator for
a period of a few weeks with no loss of activity.
The galactose acrylate (1) was dissolved in deionized water at a
concentration of 20-40% (w/v), along with the cross-linker
N,N'-Methylene-bis-acrylamide at 34% (w/w) of the monomer
concentration and the acrydine DNA at a concentration of 0.1-1 mole
% of the bis-acrylamide concentration. This procedure uses a few
nmoles of DNA for a 1 mL synthesis. The polymerization is
accomplished via a free radical polymerization, common for
formation of poly(acrylamide) gel matrixes. N,N,N',N'-tetramethyl
ethylenediamine (TEMED) and sodium persulfate are used to initiate
polymeriation. This scheme is depicted below.
##STR00002##
We have applied this technique to oligonucleotides containing 20
bases with an acrylate group on the 5' end and a fluorophore (Cy3)
on the 3' end. FIG. 2. shows the fluorescence intensity of the
immobilized DNA (circles). The intensity does not change with
repeated washings indicating the DNA is covalently immobilized. On
the other hand, when non-acrylated DNA is used (squares), the
fluorescent intensity decreases to the background level (diamonds)
after two washes. This shows that there is extremely low
non-specific absorption of target molecules to the sugar acrylate
gel. This provides the low background levels necessary for
ultrasensitive detection.
Formation of Amino-Modified Sugar Acrylate Hydrogel
Thin hydrogels (.about.100 micron thickness) were formed on glass
slides that had been functionalized with acrylic groups through the
procedure above. The galactose acrylate (1) was dissolved in
deionized water at a concentration of 2040% (w/v), along with N,N
methylene bis-acrylamide cross-linker at 3-4% (w/w) of the monomer
concentration and N-(3-aminopropyl) methacrylamide 4-5% (w/w) of
the sugar acrylate monomer concentration. The polymerization is
accomplished via a free radical polymerization using the initiators
TEMED and sodium persulfate.
In order to study the porosity of the sugar acrylate gel, we
measured the passive diffusion of fluorescently labeled beads
through a non-modified sugar acrylate hydrogel. FIG. 3. shows the
diffusion of FITC-labeled 2 micron diameter polystyrene beads
through poly(6-acryloyl-.beta.-O-methyl galactopyranoside) hydrogel
swollen in 0.25 M PBS. The gel had a 94 wt % aqueous solution
content. The curve fit indicates that at t=.infin., .about.384,000
beads will have passed through the gel into the receiving chamber.
When the experiment was done with no gel in place, at equilibrium
.about.2,110,000 beads had entered the receiving chamber. Thus,
3.84/21.1 or .about.18% of the beads that enter the gel actually
pass completely through it, and the remaining 82% become trapped,
indicating that the large pores are interconnected, and allow
significant diffusion of the 2 micron spheres through the gel
volume. The gels can be formulated to have a pore size ranging from
0.1 microns in diameter to 0.6 microns in diameter using the
original synthesis conditions described previuosly (Martin, 1998),
and by using the synthesis conditions described herein, pore sizes
of significantly greater than 2 microns in diameter can clearly be
achieved.
Linking of Oligonucleotides to Amino Sugar Gel
The amino moieties that have been linked into the gel are activated
for attachment to an aminated oligonucleotides segment using a
water soluble homobifunctional crosslinker bis(sulfosuccinimidyl)
suberate (BS.sup.3) which contains a reactive n-hydroxysuccinimide
ester (NHS-ester). The crosslinker is added to the gel under acidic
conditions (10 mM sodium phosphate, pH 6.0) at a concentration of
2.5 mM BS.sup.3 and allowed to react for 1 hour to form a stable
covalent amide bond This creates an amine reactive group on the
backbone of the gel. The entire scheme is depicted below.
##STR00003##
The amino terminated DNA is then added spot-wise to the activated
gel using a BioChip non-contact microdispensing system. The
microarrayer prints an array of oligonucleotides (900 pL per spot)
resulting in a spot diameter of 300 .mu.m and an interelement
distance of 500 .mu.m. The concentration of oligonucleotide was
from 6.25 .mu.M to 100 .mu.M. The DNA is allowed to react with the
activated substrate for 12 hours. The gel is then rinsed three
times with a 4.times. saline sodium citrate buffer solution (0.60 M
NaCl, 60 mM sodium citrate) to remove unattached DNA segments. We
have applied this technique to oligonucleotides containing 24 bases
with an amino group on the 3' end and a fluorophore (Cy3) on the 5'
end. The resulting array can then be visualized using a
conventional fluorescent array reader. FIG. 4 below shows a
photograph of a 10.times.5 array created in this manner, where the
rows are a serial dilution of the DNA. Each row contains a
replicate of ten spots, with a dilution by 2 between rows (top
row=100 .mu.M second row=50 .mu.M, third row=25 .mu.M, fourth
row=12.5 .mu.M, bottom row=6.25 .mu.M. Note that these arrays
appear approximately one-hundred times brighter relative to the
same concentration spotted onto a flat, aminosilane substrate using
the same crosslinking procedure.
Formation of Carboxylate-Modified Sugar Acrylate Hydrogel
Thin hydrogels (.about.100 micron thickness) were formed on glass
slides that had been functionalized with acrylate groups through
the procedure above. The galactose acrylate (1) was dissolved in
deionized water at a concentration of 20-40% (w/v), along with the
cross-linker N,N'-Methylene-bis-acrylamide at 3-4% (w/w) of the
monomer concentration and 2-acrylamidohydroxyacetic acid 4-5% (w/w)
of the sugar acrylate monomer concentration. The polymerization
procedure is the same as for the amino-modified hydrogel.
Linking of Oligonucleotides to Carbon Sugar Gel
Five .mu.moles of 1-ethyl-3-(3 dimethylaminopropyl)
carbodiimide-HCL (EDC) are added to the amino terminated
oligonucleotide solution at pH 7.2-7.4. The DNA/EDC solution is
then added spot-wise to the gel using a non-contact microdispensing
system. The DNA is allowed to react with the gel matrix for 12
hours at room temperature. The gel is then rinsed three times with
4.times. saline sodium citrate buffer solution to remove unattached
DNA segments. We have applied this technique to the same amino
modifed oligonucleotides described above. We arrayed these oligos
on a carboxylate-modified gel in a serial dilution starting at 25
.mu.M. FIG. 5 indicates that immobilization of the DNA is
occurring, but the fluorescent intensity is lower than observed
using the BS.sup.3 crosslinker. Note that in this case we are
starting at 1/4 the density, so the top row here should be compared
to the third row above.
Linking of Proteins to Amino Sugar Gel
An amino functionalized sugar acrylate was activated with BS.sup.3
using the procedure described above. The protein, Staphylococcal
enterotoxin B (SEB), prepared in 10 mM sodium phosphate, pH 7.4
reacts with the NHS-ester gel support Reaction of the ester with
the lysine moiety of the protein provides the final amide linkage
to the gel substrate.
Linking of Proteins to Carboxy Sugar Gel
A carboxy-functionalized sugar acrylate was activated using
carbodiimide chemistry as described above. The protein, Cy3-labeled
Staphylococcal enterotoxin B (SEB), prepared in 10 mM sodium
phosphate, pH 7.4 was allowed to react with the carboxylic acid
moiety in the presence of EDC. Reaction of the carboxylic acid
group with the primary amines of the protein provided a stable
covalent amide linkage between the protein and the gel substrate.
The SEB solution (concentration range 0.1 .mu.L to 200 .mu.g/mL)
was deposited in replicates of 15 onto the modified gel using the
BioChip microarrayer. Each printed element had 300 .mu.m spot
diameter, 900 pL print volume, and 500 .mu.m inter-element
distance. The protein modified gel slides were rinsed briefly with
PBS, pH 7.4, H.sub.2O, air dried and subsequently stored at
4.degree. C. FIG. 6. shown below indicates that we are getting
significant immobilization of the Cy5-labled SEB with the
carboxylated sugar acrylate gel.
Methods for assaying biomolecules of interest include well known
optical, fluorescence, and radioactivity means and the like,
depending on specific molecules selected for assay.
* * * * *
References